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Chapter One
Introduction
Optical Tweezers is a tool invented by ARTHUR ASHKIN in 1970. He was
motivated by a simple calculation: using a focused beam of power 1 W
striking a particle of radius of  1 wavelength we get, by conservation of
momentum, a force of  10 3 dynes, assuming the particle acts as a perfect
mirror reflecting all of the incident light momentum back on itself. Though it
is small in absolute terms, because of small mass, the acceleration imparted
is 10 5 g where g is the acceleration due to gravity! In his experiments,
Ashkin observed something even more interesting. He observed that the
particles were not only pushed by the radiation, they were drawn in to the
axis of radiation. This was accounted to be due to intensity gradient in the
laser beam. [1] This propelled the idea of optical trapping, to hold and
manipulate minute objects of micron size. Soon, Ashkin and coworkers
trapped the tobacco mosaic virus, certain bacteria and Escherichia Coli
bacteria, [2] to show experimentally that biological objects transparent to
the radiation used, could also be trapped, which led to a revolution in the
micro-studies in biology.
After the simple experiments on silica particles, experiments were performed
demonstrating atomic beam deflection [3]. Many further experiments also
revealed that atoms could also be trapped and cooled using optical tweezers
[4]. Various configurations of the lasers showed that the gradient force could
be used to levitate and hold micron size objects [5]. In recent experiments
researchers have trapped and moved the biological cell three dimensionally
[6]. They have measured elasticity of DNA by fixing one end to a substrate,
other to a dielectric bead, to hold and stretch it, with help of optical
Tweezers [7]. With the help of short pulsed lasers, along with the optical
tweezers, holding and cutting of cell membranes has been possible, making
a novel micro-surgery [8]. The amazing capability of the Optical Tweezers
was shown in the experiment of measurement of motion of Kinesin Molecule
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on a protein, which moved in nanometer steps! [9] An excellent review of all
these developments using optical tweezers is given by Ashkin. [10]
The experimental setup:
Possible experimental setup is shown below, the actual setup prepared in
this project is described in Chapter 3.
Fig 1. Showing experimental setup of optical tweezers. [11]
The laser light from a continuous wave source is expanded to cover largest
possible part of the focusing lens 100x, 1.25 Numerical Aperture. The beam
is focused onto a Polystyrene ball near the cover slip. The light of condenser
from below is split into two paths, one going to Eyepiece and other to CCD
for recording. The setup is basically that of inverted microscope.
The setup can be modified in various ways. For position measurement of the
particle, for example, a Quadrant Photo detector could be used in place of
2
CCD camera. For vibrations of a Bead attached to DNA molecule, it could be
shined with another laser light and scattered light be collected on a
photodiode in place of CCD camera.
For advanced experiments like study of elasticity of DNA, the setup table
needs to be isolated from the vibrations by using a floating table.
Principles of Working:
Basic principle of working of Optical tweezers is the radiation pressure. The
particles receive a back kick from transmitted radiation. Hence we need
material with high refractive index and transparency. Such a material bends
the light more and hence causing larger change in momentum. Particles
with lower relative refractive index than the medium, like air bubbles are
seen to be repelled from the trap. [1]
The analysis of working can be done in two ways,
First, for particle size << , using electrodynamics equations to calculate the
power exerted on a dielectric sphere.
Second, in the regime where size >> , where ray optics holds.
First case is large subject matter of [12, 13 and 14] which we will not go
into here, but we will discuss the ray optic analysis to get the understanding
of origin of trapping force.
Qualitative as well as quantitative analysis of this was done by Ashkin and
others in [15, 16].
Considering the cases when particle is in different
positions relative to the focus of the laser light, analysis of forces shows that
the net resultant force is always directed to this focus, showing that the trap
is stable. The weakest direction of the trap is the one after focus in the
direction of propagation of laser. The analysis is done by considering few
prototype rays as follows:
3
Fig 2: Comparison of gradient force Vs. Scattering force.
The two forces arising are due to reflected and refracted rays. The reflected
rays tend to scatter the particle, the refracted rays give back kick to the
particle required for trapping, and this force is the gradient force. For high
numerical apertures, i.e. for high focusing of radiation, the gradient force
can be made to overcome the scattering force resulting into trapping of
particles, as shown in the figure above. It can also be seen to be dominant
force for other positions of particle relative to focus as follows:
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Fig 3. Force on the particle is always directed to the point of focus of the
laser beam.
It can be shown that the force is independent of radius of the particle in this
regime, where size>>. For the Mie regime where size << , the force varies
as r 3 . So for particle sizes in between, i.e. size  , we expect the variation
to be between r 0 and r 3 . [15]
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The form of the force calculated by considering the geometry, numerical
aperture, power and refractive index of particles is F  Q .
nP
, where Q is a
c
dimensionless factor, n is the refractive index, P is the power of laser and c
is the speed of light. For TEM 00 mode and numerical aperture of 1.25, Q is
0.27. For this value of Q, in the weakest direction of the trap, a spherically
shaped motile living organism must exert the force of approximately 1.2
micro dynes, for power of laser 10 mW. This implies that a motile organism
10 micron in diameter which is capable of propelling itself through water at
a speed of 128 micron/sec will be just able to escape the trap, in its weakest
direction. [15]
The trapping is more dependent on geometry than on laser power. Higher
laser power can cause local heating harmful to the biological species.
Trapping energies of non-motile samples are just of the order of kT, the
thermal motion energy provided by the surrounding water to the specimen.
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Chapter Two
Background
Since the discovery of Optical trapping by Ashkin in 1970s, there has been a
spur of activity in application of optical tweezers to biological studies, as
mentioned in the introduction. Following are some of the prototype examples
of works experimentalists have carried all over the world, using the optical
tweezer as a tool. These examples also describe the essential techniques
involved in experiments using optical tweezers.
I. Work on Optical Trapping and Manipulation of Viruses and Bacteria:
Soon after the discovery of trapping of micron sized polystyrene latex
colloidal
particles
and
submicron
size
silica
spheres,
Ashkin
and
researchers experimented trapping of colloidal tobacco mosaic virus (TMV).
[2] Tobacco mosaic virus is a rugged, rod like protein that traps easily and
orients itself within the trap. Interesting side-benefit of this experiment was
the observation that some increasing number of strange, relatively large,
apparently self-propagating particles. Suspecting bacterial contamination,
they combined the trap with a high resolution microscope. This confirmed
the trapping of live motile bacteria and their subsequent “opticulation”
(death by light).
This was because of strong absorption of green light by
biological species. Later experiments with Infrared yttrium/aluminum
garnet laser showed drastic changes. It became possible to hold E-coli
bacteria and yeast cells for hours in isolation and observe cell reproduction
within the trap. Damage-free trapping of pigmented red blood cells, green
plant cells, and algae was also shown. These and some more experiments on
internal cell manipulation marked the beginning of the new field of optical
trapping in biology.
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The schematic of the experimental setup is as shown below:
Fig 4: The 90 degree scattering from trapped particles can be viewed visually
through a beam splitter (S) with a microscope (M) or recorded using a photodetector (D). [2]
Main components used were:
Spatially filtered Ar green laser of wavelength 5145 A0 , with power varied
from 100 to 300 mW. The Gaussian beam was focused to spot diameter of
0.6microns using an objective lens of water immersion type with numerical
aperture of 1.25.
For identification of bacteria, microscope with 800X is used.
Procedure and discussion:
The sample preparation: The sample can be prepared in mono-disperse
colloidal suspension in water at high concentrations. TMV is a protein of
cylindrical shape, with diameter 200 A0 and length of 3200 A0 . It has
negative charge in solution and an index of refraction of about 1.57. They
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align themselves in parallel arrays in dense aqueous solutions. Somewhat
diluted samples of these kind were used in the setup shown above, which
was also used for trapping of silica and other particle spheres.
Observation of trapping: Trapping is observed at laser powers of about 100
to 300 mW. The capture of a virus manifests itself as a sudden increase in
90 degree scattered intensity. The bacteria could be captured into the trap
more easily, as they are less motile, at powers of 3-6 mW. These could be
moved across transversely to capture many more bacteria. The trapping of
many bacteria is possible with rapid motion in transverse direction, because
the forces in transverse are much stronger than those in the axial. Also, the
forward direction force in the axial direction is stronger than the reverse
direction, so a rapid upward motion can result in escape of particles. At even
lower powers, 1-3 mW, a different trapping mode could be observed, in
which bacteria were trapped against the bottom surface of the cover slide. In
this weakest direction of trapping force, the mechanical force is provided by
the slide. It is still possible to move the particles transversely over the
surface, because transverse forces remain quite strong even at low powers.
Trapping of E-coli bacteria was even easier because of their less motility,
and could be captured and manipulated rapidly on the surface and in the
bulk with powers as low as fraction of an mW.
Size determination: Analysis of scattered light intensity, its angular
dependence gives the information about the size, shape and orientation of
the trapped viruses. Polystyrene latex spheres of known size are used to
calibrate the intensity vs. size. The volume computed after averaging several
trapped particles, gives, an effective volume of (450 A0 ) 3 . This corresponds to
a cylinder 200 A0 in diameter and 3100 A0 long, which is quite close to the
volume of TMV. From this it can also be concluded that single TMV particles
are being trapped. Occasional trapping of more than one virus results into a
dip in photo-detector intensity.
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Opticulation: the TMV did not suffer any changes in size, and could be
trapped for several minutes without optical damage, for powers of the order
of 120 mW. The reason for this stability in visible range is that these
particles have strong absorption in UV. On the contrast, bacteria which
could be trapped very easily at low powers of the order of few mW, were
killed at 100 mW.
II. Experiments in fertilization:
Experiments were performed with tweezers to manipulate live sperm cells in
three dimensions [18,19] and to measure their swimming forces [20].
Applications of tweezers with short pulsed lasers called “scissors” to all
optical in vitro fertilization are being considered [21]. UV drilling of channels
in zona ellucida of oocytes was shown to assist sperm penetration [21].
Tweezers was used to insert selected sperm into channels to effect
fertilization [22, 23]. However, fertilization efficiency and questions of
possible genetic damage must be further studied. Important experiments by
Bern’s
group measured the effects of the wavelength on optical damage
processes in sperm and in other contexts using tunable Ti sapphires lasers
[24].
III. Work on DNA flexibility using Backscattering from a tethered bead
as a probe:
For polymer based biosensors, DNA, being a good mechanically flexible
molecule could be used. Shivashankar et al, [7] used optical tweezer to hold
the bead attached to one end of a DNA, other end of which is attached to the
glass plate. The schematic of the setup is as follows:
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Fig 5: Inset shows Bright field image of a DNA tethered bead in the optical
trap and the backscattered light image of the same bead illuminated with
the red laser. Both the images are visualized using a CCD camera.
Main components used for the experiment are:
The optical trapping laser used is near infrared 830 nm, 150 mW maximum
power laser.
Infinity corrected objective lens (Zeiss Neoflar 100 X, 1.3 NA, oil immersion).
Red laser to scatter light from bead, 8mW, 633 nm.
Quadrant detector (UDT, Spot 4D.)
Procedure and discussion:
The red laser light scattered from the bead gives horizontal position of the
bead using the differential output of the quadrant detector. Total sum at the
quadrant detector gives the changes in the vertical position.
The delicacy of the experiment lies in the precise motion of the piezoelectrically controlled stage. The minimum step size of the stage is 10 nm.
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Force calibration is done using the Stokes drag method, by measuring the
translational velocity of the stage for which a 3 micron un-tethered bead
escapes from the trap. For the 50 mW trapping power, the maximum
trapping force is of the order of 3.7 picoN.
Initial adjustments of the quadrant detector are done using an immobile
bead. It is then raised to 3 micron height. The λ-DNA used is of length 15
microns. Keeping the bead position constant, cover slip is moved in x
direction to stretch the molecule to maximum length. The motion of the
bead in parabolic trap of optical tweezer indicates the freedom for its
vibration as constrained by the DNA molecule. So as the strand of DNA is
stretched, this motion gets restricted and can be seen from the histograms
of the x-y positions obtained from the quadrant detector. Since the
stretching of the DNA is along the x-direction, there is asymmetry visible in
the fluctuations of the bead; they are reduced in the x-direction.
The observed measurements of position are found to be fitting in the
theoretical analysis based on a potential for DNA. In some more
experiments, by adding a protein called Rec A, which polymerizes the DNA
molecule, the researchers have found that the polymerization extends the
DNA molecule beyond its contour length. Thus this technique has provided
a direct measurement of DNA unfolding and kinetics of binding to Rec A
protein.
IV. Quantitative measurements of Force and Displacement using an
Optical Trap.
R. M. Simmons and coworkers combined a single beam gradient optical trap
with a high resolution photodiode position detector to show that an optical
trap can be used to make quantitative measurements of nanometer
displacements and pico-newton forces with millisecond resolution[25]. When
an external force is applied to a micron sized bead held by an optical trap,
the bead is displaced from the center of the trap by an amount proportional
to applied force. When the applied force is changed rapidly, the rise time of
the displacement is on the millisecond timescale, and thus a trapped bead
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can be used as a force transducer. The performance was enhanced by a
feedback circuit so that the position of the trap moves by means of an
acousto-optic modulator to exert a force equal and opposite to the external
force applied to the bead. The parameters of the trapped bead such as
stiffness and response time as a function of bead diameter, bead position
within the trap and laser beam power, were compared with recent ray optic
calculation of the forces on trapped beads [15].
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Chapter Three
Making of the Experimental Setup:
Testing of the Biological Microscope:
Schematic diagram of the Microscope used for the setup is as shown below:
Eyepiece
Objective Lens,
could be changed
from 8X to 40X
and 90X
Sample table
Contrast
Adjustment
3-D movable
mirror
Fig 6: Schematic diagram of the microscope.
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Study of the functioning of microscope:
The microscope has three objective lenses and one eyepiece.
Any of the
three objective lenses can be used to observe the sample, by rotating the
holder of these lenses. The light gathered on the plane of the sample can
also be varied, to adjust the contrast, using a focusing lens provided
beneath the sample. The mirror has three-dimensional rotation possible, so
that the place of the illuminating lamp, a tube light in this case, doesn’t
affect, and the light could be directed in the sample. This facility of rotating
mirror proved to be very useful when we guided the laser light and visible
light alternatively into the sample. The vertical focusing of eyepiece can be
adjusted with a very high precision, the least count of the motion being 2
micron.
Troubleshooting the microscope:
To begin with, cleaning of all the objective lenses and mirrors was done
using methanol with the help of cotton plugs and tissue paper. We tested
the microscope first of all with dust particles. The microscope has three
magnifications, 8 X, 40X, and 90 X. The dust particles could be seen easily
with 8X, but they lacked the irregularity on a micron scale, so could not be
observed well under the 40X and 90X lens. So we looked out for two more
samples: smeared out RBC slide, onion cells.
RBCs have suitable size for the large magnifications of 40X and 90X, and
could be seen as a smeared out web. While viewing the sample under 90X
magnification, a problem was faced that the plane of the focus could not be
reached while lowering the objective. It was realized that the sample was
mounted upside down, thus RBCs were on the other side of the glass slide.
This restricted viewing of RBCs using 90X, though they were seen through
40X. Upon reversal of glass slide, RBCs could be seen using 90X also. This
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showed that 90X objective has a very small focal length, of the size of glass
slide, i.e. less than 2mm.
A thin layer of onion was peeled off from the skin of onion, to make a slide of
onion tissue. In 8X, linear arrays of cells were seen. In 40X, few long
parallelogram like onion cells were seen. In 90X, we saw one single
magnified onion cell, surrounded by other. The cell walls between the cells
could be clearly seen.
As preparation for the further work on trapping, bacteria were observed
under the same microscope. The bacteria could be observed clearly in 40X.
Modification in the microscope to suit to Optical Tweezer setup:
We need to inject the laser light into the objective of the microscope to focus
the laser and trap the bacteria or RBCs. For this purpose, the top viewing
part of the microscope was removed and laser light was injected vertically
from the above. For viewing the sample, we introduced a glass slide in the
path of laser light at 45 degrees, and saw the reflected light image through
the eyepiece, after arranging the eyepiece at 90 degrees to the laser light.
At first, it was very difficult to get the alignment of the glass slide and the
eyepiece. So laser light was used from below, instead of above, to align the
eyepiece. Then the laser light was replaced by usual illumination, to view the
sample of smeared out RBCs slide.
The slide of RBCs could be seen in 8X and 40X, though with much lower
intensity and clarity than before. The RBCs could not be seen through the
90X lens. The reason turned out to be the low light gathering aperture of the
90X lens. Also the low reflection coefficient of glass, 4%, resulted in loss of
image.
16
In these attempts, and in the experiments that followed, to adjust the
eyepiece accurately at 90degree, and align for the reflected light, a mirror
was used in place of the reflecting glass, to see image in the eyepiece. Mirror
provided large reflection in comparison to glass slide, and could be used for
the alignment and positioning of eyepiece very effectively. Also, the path
length between the sample and eyepiece was kept more or less same as in
the microscope, so as to achieve proper focusing.
To overcome the problem of low reflected intensity of light, we chose to
increase the intensity of illuminating light, using bulbs. The yellow bulb of
100 W got heated up very quickly, and also was too bright, and the sample
would not be visible because of lack of contrast. The less intense milky bulb
of 100 W also showed no contrast and the whole image used to get wiped
out. Thus the option of using bulb was ruled out. It was thought of that one
laser could be used to illuminate the sample and other to be focused as the
Tweezer, but this too was ruled out because in the laser light, the details of
sample were difficult to figure out.
We tried carbon coating the glass slide to improve the reflection of image of
the sample. The coating was done using the flame of a candle. This showed
the images of the sample in 90X. But this method had the drawback that
the laser light was totally scattered and lost its intensity after passing
through the carbon coated glass slide. Hence a reduction of coating was
tried out, by moving the glass slide only once over the candle. But this
showed the image with less clarity and still the laser light was reduced in
intensity after scattering.
Finally, availability of a partially reflecting glass solved the problem of
viewing the sample at the same time passing the laser light. This glass
seemed to have around 10-12% reflection, and image of sample could be
seen in 90X magnification also.
17
In other experiments on optical tweezers, experimentalists have used the
dichromatic mirror, which reflects the illuminating wavelength, and
transmits the laser light. In our setup, we didn’t need to use of a
dichromatic mirror.
Need of crossed polarizer:
After availability of the partially reflecting glass, we used laser light to shine
the sample vertically from the top. Initially we had anticipated that only the
sample will reflect the laser light, and hence laser light would be very small
in intensity on the eyepiece. But this assumption turned out to be wrong,
and the upper part of the lens reflected large amount of light, which got
reflected from the partial reflecting glass slide, and destroyed the image in
the eyepiece. Hence, to cut the laser light, we used crossed polarizeranalyzer pair, polarizer immediately after the laser light and analyzer before
eyepiece. This allowed the un-polarized visible light to pass through, and the
laser light to get blocked.
Glare of the laser light from the sample:
After removing the reflection of laser light from the surface of the lens, using
crossed polarizers, it was still observed that the sample with the cover slip
shines back some glare of the laser light on the partially reflecting plate and
hence enters in the eyepiece. This glare was removed by selectively focusing
on the image in a small region of reflected light.
Thus the final setup took this shape:
18
He Ne Laser
Polarizer
Partial
Reflector
Analyzer
Eyepiece
Fig 7: Final Setup for Optical Tweezer.
19
Chapter 4: Experimental Observations:
Bacteria:
A sample of bacteria subtilis was obtained from the Bio Technology Center.
The bacteria were cultured overnight, before using them for the experiments.
The bacteria survive at room temperatures for and hour or two, and need to
be preserved in the refrigerator, where they could be stored without decay
for a day.
A drop of the sample of bacteria is taken on a glass slide and covered with a
cover slip. The sample is then viewed under the microscope. The objective
lens often touches the cover slip forcing the liquid between to move out, in
which case the slide is to be again. The solution of the bacteria dries out
quickly, so it has to be viewed in a short while.
These bacteria are like long ropes, and they constantly keep moving in the
field of view and changing their shape. They appear on the turbid
background once in a while and disappear in the same, since the motion is
not confined to the single plane.
RBCs:
Red Blood cells were isolated from a freshly collected blood sample at the
school of Bio Medical Engineering. An anticoagulant solution was added to
stop the blood from coagulating. The RBC sample was diluted in the saline
water solution, to decrease the density of RBCs, so that they do not
congregate in the field of view, and also to provide a medium for the RBCs to
float around.
A drop of this sample is taken on the glass slide and covered with the cover
slip. For initial observations, the sample turned out to be densely packed set
of RBCs. So it was further diluted to decrease the RBCs in field of view. This
also allowed the motion of RBCs. But the group of RBCs moved together in a
direction and slowed down until they reached the standstill, in contrast to
the expectation that they would move independent of each other, due to
thermal motion.
20
Both the samples have been viewed properly in 40X objective lens, but the
90X lens could not be used because of very short focal length.
21
Chapter Five: Conclusion
An experiment has been set up for the optical trapping of
bacteria, RBCs and other biological samples. The problem lies in
viewing the samples through 90X magnification. Some other
samples also need to be considered for trapping, as RBCs stick
up to the glass slide and cover slip. The bacteria sample has a
very small lifetime. These difficulties will be overcome by the time
of presentation and the final results will be presented.
This Project has introduced me to the exciting research in the
field of Optical Tweezers. It has given me a very good training in
literature survey, planning and development of experimental
setup.
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References:
[1] First Paper: “Acceleration and Trapping of particles by radiation
pressure”, A.Ashkin, Phys. Rev. Lett.1970 (156-159).
[2] "Optical Trapping and Manipulation of Viruses and Bacteria", Ashkin, A.;
Dziedzic, J.M., Science 235, (4795) pp 1517-20 (1987).
[3] “Atomic-Beam deflection by resonance radiation pressure”, Ashkin, A.
Phys. Rev. Lett., Vol.25, No. 19, 9 Nov.1970.
[4] Bjorkholm, J.E. et al,(1978) Phys. Rev. Lett. 41, 1361-1364.
[5] Ashkin A.,Dziedzic J.M.(1971) Appl.Phys.Lett. 19, 283-285.
[6] Ashkin A.,Dziedzic J.M.(1989) Proc. Natl. Acad. Sci. USA 86.7914-7918.
[7] Backscattering from a tethered bead as a probe of DNA flexibility”,
Shivashankar et al, Appl. Phys. Lett. Vol. 73, No. 3, 20 July 1998, 291-293.
[8] Steubing, R.W., et al, (1991) Cytometry 12, 492-496.
[9] Block,S.M.,et al, (1990) Nature (London) 348,346-348.
[10] Excellent Review paper: “Optical Trapping and manipulation of neutral
particles using lasers”, A. Ashkin, Proc. Natl. Acad. Sci. USA Vol. 94, pp.
4853-4860, May 1997.
Available online: http://www.dfi.aau.dk/~balling/alp/ashkin.pdf
[11] http://atom.harvard.edu/tweezer - shows how to build a tweezer and
troubleshoot it.
[12] “Internal and near-surface electromagnetic fields for a spherical particle
irradiated by a focused laser beam”, J.P. Barton et al, J. Appl. Phys. 64 (4),
15 August 1988. (1632-1639)
[13]
“Fifth
order
corrected
electromagnetic
field
components
for
a
fundamental Gaussian beam”, J.P. Barton and D.R. Alexander, J. Appl.
Phys. 66(7), 1 October 1989. (2800-2802)
[14] “Theoretical determination of net radiation force and torque for a
spherical particle illuminated by a focused laser beam”, J.P. Barton et al, J.
Appl. Phys. 66(10), 15 November 1989. (4594-4602)
[15] “Forces of a single beam gradient laser trap on a dielectric sphere in the
ray optics regime”, A. Ashkin, Biophys. J. Vol. 61, Feb. 1992, 569-582.
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[16] “Observation of a single beam gradient trap force optical trap for
dielectric particles”, A. Ashkin et al, Optics Lett. 11:288-290, 1986.
[17] “Optical Tweezers: A new Tool for Biophysics”,Steven M.Block,
Noninvasive Techniques in cell Biology:375-402 © 1990,Wiley-liss Inc.
[18] Tadir, Y., et al, (1989)Fertil. Steril. 52, 870-873.
[19] Colon, J. M. et al, (1992), Fertil. Steril. 57, 695-698.
[20] Bonder,E.M. et al, (1990), J. Cell Biol. 111,421a.
[21] Tadir,Y., et al,(1991), Hum.Reprod.6,1011-1016.
[22] Schutze,K.,et al,(1994),Fertil.Steril.61,783-786.
[23] Clement-Senggewald,A., et al, J.Asst.REprod.Genet.13,259-265.
[24] Liang, H., et al, Biophys.J.70, 1529-1533.
[25] “Quantitative Measurements of Force and Displacement Using an
Optical Trap”, R. M. Simmons et al, Biophysical J., Vol.70, April 1996, 18131822.
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